Differential localization of 5-hydroxytryptamine3 and 5-hydroxytryptamine4 receptors in the human rectum

The functions of the 5-hydroxytryptamine3 (5-HT3) and 5-hydroxytryptamine4 (5-HT4) receptors in gastrointestinal tract are complex depending on the species and anatomical regions, and the localization of these receptors in the human rectum was unclear. We examined the localization of the 5-HT3 and 5-HT4 receptors in human rectum by in vitro receptor autoradiography using [125I](S)iodozacopride and [125I] SB207710 as a ligand, respectively. Specific [125I](S)iodozacopride binding sites were clearly evident in the myenteric plexus, whereas, low levels of [125I]SB207710 binding sites were distributed over the muscle but not to the myenteric plexus. The 5-HT3 receptor located on the myenteric plexus and the 5-HT4 receptor on the smooth muscle may participate in contractility and relaxation of human rectum, respectively.     

Optimal antigen localization in human tissues using aldehyde-fixed plastic-embedded sections

Although the utility of antigen labeling techniques in frozen tissues is well known, it is generally acknowledged that an improvement in morphologic preservation is desirable. Conventionally processed paraffin-embedded tissues are limited in the range of antigens that can be detected and newer plastic embedding techniques have been even more restricted. By using cold (4 degrees C) processing and limited fixation a wide range of antigens (including T and B markers) has been demonstrated in 2 mu plastic sections. The morphologic preservation and antigen localization are superior to other techniques. The combination of precise antigen localization and excellent morphologic preservation should expand the diagnostic and investigative uses of immunohistology.     

Identification of catecholamines in the rat brain and mollusk tissues using semithin slices and retrograde staining

The suggested technique allows revealing the transport-specific dye (primulin) and catecholamine fluorescence simultaneously in the same cell of brain. Intense fluorescence is observed when brain tissue is quickly dehydrated and embedded in the epoxy resin. The same method is suggested for the identification of catecholamines in the embryonal and juvenile tissues of gastropod Lymnaea stagnalis (Mollusca Pulmonata) without using of primulin dye.     

Unconventional application of standard light and electron immunocytochemical analysis to aldehyde-fixed, araldite-embedded tissues

A simple procedure for the immunocytochemical analysis of glutaraldehyde/formaldehyde-fixed, Araldite- or Epon-embedded tissues by either light or electron microscopy is presented. Retention of immunoreactive antigen in deplasticized sections was achieved by use of a low concentration of glutaraldehyde in the fixative in combination with a seldom-used plastic solvent. This protocol produced good ultrastructural preservation in tissues and large, high-quality, 2-micrometers thick, plastic-free sections. These semithin sections provided a level of structural and antigenic preservation, image resolution, and labeling intensity that surpassed all other conventional sectioning methods used for immunocytochemistry. The capacity to use a single tissue sample in studies designed for light and electron immunocytochemistry, in conjunction with existing autoradiographic and cytochemical techniques, makes this a very desirable method for routine tissue preparation in research and clinical applications.     

Histochemical studies on the distribution of catecholamines and 5-hydroxytryptamine after intraventricular injections

Summary Using the histochemical method for the demonstration of DA, NA and 5-HT it has been possible to demonstrate, in reserpine treated rats, that intraventricularly administered DA, NA, -methyl-DA and -methyl-NA in doses of 1–2 g are specifically taken up by the parts of the DA and NA neurons lying close to the ventricles and the subarachnoidal space. The distribution of this uptake is described in detail. No uptake and accumulation of DA and NA was observed unless the monoamineoxidase had been inhibited whereas the -methylated compounds which are resistant to monoamineoxidase accumulated without monoamineoxidase inhibition. Intraventricularly administered 5-HT was specifically taken up and accumulated in the 5-HT neurons within the same zone provided that monoamineoxidase had been inhibited. The distribution of this uptake is described in detail. After high doses of CA (5–10 g) these amines accumulated to some extent also in the 5-HT neurons while no such accumulation was observed in the CA neurons after high doses of 5-HT. Thus, the present results indicate that there exists a specific reserpine-resistant, amine-concentrating mechanism at the nerve cell membrane of CA and 5-HT neurons. In areas where the exogenous amine concentrations probably were high there also occurred an accumulation of DA and NA in the CA neurons although the monoamineoxidase was not inhibited. Finally, in a certain area of the hypothalamus, CA was found to accumulate even after low doses (1–2 g), in nerve cell bodies which probably normally do not contain CA.This study was supported by a research grant from the Swedish Medical Research Council (12x-715-03) and by grants from M. Bergwalls stiftelse and C. Nathorsts stiftelse.     

Specificity of the glutaraldehyde silver technique for catecholamines and related compounds

Summary Studies regarding the specificity of glutaraldehyde-silver technique showed that this technique, used as described previously is positive for noradrenalin, dopamine, melanin and lipofuscin. Adrenalin and 5-hydroxytryptamin storing cell produce negative results.Light microscopy does not reveal 5-hydroxytryptamin containing cells even following prolonged (30 minutes) silver treatment, but a fine silver precipitate appears under these conditions in 5-hydroxytryptamin containing granules when the material is observed under the electron microscopy.Dopamine is not revealed by light microscopy because the silver precipitate is very fine and scattered in the granule but it is easily seen by electron microscopy.Abbrevations used DA dopamine - NA noradrenalin - A adrenalin - 5-HT 5-hydroxytrdytamin - GA glutaraldehyde - S silver     

Binding of catecholamines and related compounds to proteins after formaldehyde gas treatment

Summary Primary catecholamines e.g. dopamine and noradrenaline easily condense with formaldehyde in both tissues and model protein layers, yielding strongly fluorescent 3,4-dihydroisoquinolines. This reaction can be used for the histochemical demonstration of biogenic monoamines according to the method of Falck and Hillarp. The investigation reported presents data which suggest that the isoquinolines formed are mainly enclosed in or adsorbed on the network formed by the formaldehyde tanning of the protein. The products seem not to be chemically bound. No difference was found in the binding of the different biogenic catecholamines. The degree of binding can be reinforced by prolonged formaldehyde gas treatment and the use of more humid gas, which might prove useful when this binding method is employed for the autoradiography of catecholamines and 5-hydroxytryptamine. The possibilities of chemical binding are discussed.     

Stability and biological activity of catecholamines and 5-hydroxytryptamine immobilized to sepharose and glass beads.

[14C]Noradrenaline, [14C]adrenaline, and 5-[14C]hydroxytryptamine were complexed to several types of Sepharose derivatives through an amide or a diazo linkage. Complexes were also made with arylamine glass beads through a diazo linkage. After the reaction the complexes were extensively washed with distilled water or dilute HCl or both, plus other solvents. Radioactivity appeared in the storage solutions and the amount increased with time. When initial low levels of release were achieved by extensive washing, the addition of a different solvent produced a marked increase in release. The biological activity of these complexes was tested on isolated rabbit aortic strips and rat fundus strips. All complexes caused the tissues to contract. The biological activity, however, closely paralleled the release of the ligands from the complexes and the release of ligand was demonstrated through the use of oil immersion and dialysis sac. These data do not support the recent claims that catecholamines bound to glass beads exert their pharmacological effects as covalently bound complexes on isolated tissue. The lack of stability of these complexes indicates the need for caution in drawing conclusions as to the site or the mechanism of action of immobilized catecholamines and other drugs.     

Differential effects of mercurial compounds on excitable tissues.

Sarcoplasmic reticulum (SR), Ca2+ plus Mg2+-ATPase, and Ca2+-ionophore were obtained from white rabbit skeletal muscles. Methylmercury inhibited the Ca2+ plus Mg2+-ATPase and Ca2+-transport but had no effect on the Ca2+-ionophore. Mercuric chloride inhibited all three functions (i.e., ATPase, transport and ionophoric activity). The mechanism of HgCl2 inhibition of the Ca2+-ionophore was by competition with Ca2+ for Ca2+-ionophoric site whereas its inhibition of the enzyme and Ca2+-transport was due to the blockage of essential sulfhydryl (--SH) groups. Ca2+ plus Mg2+-ATPase and Ca2+-transport were more sensitive to methylmercury than to HgCl2. Acetylcholine receptor (AChR) was obtained for the electric organ of T. californica. Methylmercury inhibited the ACh binding to AChR WITH Ki = 5.7 - 10(-6) M. This effect was not due to mercuric ion alone since mercuric chloride up to 10(-4) M did not affect ACh binding to AChR. It is concluded that: the Ca2+ plus Mg2+-ATPase and Ca2+-transport contain --SH groups essential for their activity, and that the two functions are tightly coupled; the Ca2+-ionophore contains no --SH groups essential for its activity; CH3HgCl inhibition of Ca2+ plus Mg2+-ATPase and Ca2+-transport is partly due to its reactivity with --SH groups in hydrophobic environment; the Ca2+-transport is inhibited by HgCl2 through two processes, one which is the blockage of --SH groups and another which is the inhibition of the Ca2+-ionophoric site; and the inhibition of ACh binding to AChR is due to the blockage of --SH groups in hydrophobic environment, which is inaccessible to Hg2+. Our data present for the first time a molecular basis for the myopathy associated with mercurial compounds toxicity.